Abstract

Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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Detection of phosphorylated and SUMOylated…

Fig. 1.

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Detection of phosphorylated and SUMOylated forms of Sox9 in the developing chick neural…

SUMOylation of Sox9 depends on phosphorylation. (A) SUMO conjugation occurs in WT-Sox9, Sox9S64A, and Sox9S181A but not in Sox9S64A,S181A mutants. Addition of CA-PKA does not rescue SUMOylation of the Sox9S64A,S181 mutant. (B) Ectopic expression of the non-SUMOylatable form of Sox9 in the developing chick neural tube is detected by anti-Sox9pS181. (Scale bar, 100 μm.) (C) SUMOylated Sox9 protein is detected in embryos transfected with WT-Sox9 or Sox9-SA mutant. (D) SUMOylation of Sox9 is enhanced by CA-PKA and markedly diminished by DN-PKA without significantly altering the levels of phosphorylated form of Sox9 compared with WT-Sox9. (E) Western blots showing similar levels of SUMOylation in both WT-Sox9 and phosphomimetic mutant. (F) Reduced amount of UBC9 is observed in IP fraction from Sox9S64A,S181A compared with the WT-Sox9. (G) V5-Snail2 and V5-UBC9 proteins are detected in the IP fraction of Sox9S64D,S181D transfected embryos. Input represents 1% of IP.

Sox9 phosphorylation is required for trunk neural crest delamination. (A) Dorsal view of the trunk neural tubes electroporated with the indicated constructs 24 hpt showing GFP-positive emigrating NCCs. (Scale bar, 50 µm.) (B) Transverse sections of electroporated embryos from A showing robust ectopic induction of HNK-1 in the transfected neural tube by each construct. WT-Sox9–, Sox9S64D,S181D-, or Sox9K61R,K254R,K376R-expressing cells are observed delaminating from the pial surface of the dorsal neural tube where laminin is lost (white arrowheads), whereas SOX9S64A,S181A-transfected cells still remained in the neuroepithelium without disrupting basement membrane (yellow arrow). (Scale bar, 50 µm.) (C) FoxD3 expression remains unaltered in Sox9 electroporated embryo at 36 hpt, whereas reduced FoxD3 expression in emigrating neural crest cells is observed on the transfected side of the neural tube (Left) with Sox9S64A,S181A compared with the untransfected side. (Scale bar, 100 μm.) (D) Electroporation of Sox9-MO into the right side of the embryo results in most of the transfected cells remain in the neural tube with a marked reduction of the amount of FoxD3-positive migrating NCCs compared with the untransfected side or with the Ctrl-MO–treated embryo where NC emigration is observed (white arrows). Coelectroporation of Sox9-MO plus WT-Sox9 restores NC emigration (white arrows) together with the amount of FoxD3-positive migrating NCCs, whereas coelectroporation with Sox9S64A,S181A fails to rescue the loss-of-function phenotype with a drastic reduced amount of FoxD3-positive migrating NCCs. (E) The number of emigrating GFP+ve cells expressing HNK-1 from the transfected side of the neural tube with the indicated constructs. Each bar represents an average of emigrating GFP+ve cells expressing HNK-1 from three to five well-transfected embryos with each analyzing 10–15 sections. *P < 0.01; **P < 0.001; ***P < 0.05.

Fig. 4.

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Sox9 phosphorylation is required for…

Fig. 4.

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Sox9 phosphorylation is required for cooperating with Snail2 to induce an EMT. (…

Fig. 4.

Sox9 phosphorylation is required for cooperating with Snail2 to induce an EMT. (A) Embryos transfected with Sox9/Snail2; SOX9S64D,S181D/Snail2; SOX9K61R,K254R,K376R/Snail2 result in NC delamination in the intermediate and ventral extent of the neural tube where expression of basal laminin and apical PKC are lost (white arrows). However, most of the Sox9S64A,S181A/Snail2-transfected cells remain in the neuroepithelium without disrupting basal laminin and apical PKC expression. (B) The amount of Snail2 protein presence in the IP fraction of Sox9K61R,K254R,K376R transfected embryos is less than that of WT-Sox9. No Snail2 protein is detected in the IP fraction of Sox9S64A,S181A-transfected embryos. (C) Schematic of WT-Sox9 or Sox9S64A,S181A-Snail2 tethered construct. Horizontal white line in the Insets indicate the plane of sectioning of the transfected neural tube with WT-Sox9-Snail2 or Sox9S64A,S181A-Snail2. Both tethered constructs can induce ectopic HNK-1 expression. Cells expressing Sox9-Snail2 emigrate from the dorsal, medial, and ventral extent of the neural tube (yellow arrows) with loss of basal laminin and aPKC expression. In contrast, the majority of Sox9S64A,S181A-Snail2-expressing cells remain in the neuroepithelium without disrupting laminin and aPKC expression. (Scale bars, 100 µm.)

BMP and canonical Wnt signaling regulates phosphorylation and SUMOylation of Sox9. (A) Sox9 expression was examined on electroporated embryos with the indicated constructs followed by transverse sections at the level indicated by horizontal black lines, and GFP immunofluorescence was performed to mark the transfected cells. (Inset) Region of transfection with β-cat-EnR in the dorsal neural fold where Sox9 transcripts are detected. Immunofluorescence on transverse sections of electroporated embryos showing expression of Sox9 and Laminin proteins remain intact. (Scale bar, 50 µm.) (B) WT-Sox9 is phosphorylated and SUMOylated, but both modifications are inhibited in the presence of Smad6. (C) Western blots showing an increased amount of phosphorylated and SUMOylated forms of Sox9 in the presence of CA-BMPR-1A compared with the WT-Sox9 alone. (D) Activation and inhibition of canonical Wnt signaling by TCF-VP16 and β-catenin-EnR promotes and abolishes Sox9 phosphorylation and SUMOylation, respectively. (E) Levels of phosphorylated and SUMOylated forms of Sox9 in the presence of TCF-VP16 or β-catenin-EnR by Western blot were quantified compared with the modified forms of WT-Sox9 alone. *P < 0.05; **P < 0.01. (F) Electroporation of CA-BMPR-1A promotes NC delamination from the dorsal neural tube. Coelectroporation of Sox9S64A,S181A plus CA-BMPR-1A results in ectopic HNK-1 expression, but transfected cells remain in the neuroepithelium without disrupting laminin expression in the basement membrane (white arrow). (Scale bar, 100 µm.) (G) Schematic diagram showing the transcriptional outputs of BMP and/or Wnt signaling can induce Sox9 phosphorylation, which facilitates SUMOylation partly through interaction with UBC9 and both modifications cooperate with Snail2 to trigger NC delamination.